一种康普瑞汀衍生物及其制备方法和应用
阅读说明：本技术 一种康普瑞汀衍生物及其制备方法和应用 (Combretastatin derivative and preparation method and application thereof ) 是由 黄晓超 陈远航 刘执坤 喻春皓 杨勇 于 2021-07-14 设计创作，主要内容包括：本发明公开了一种康普瑞汀衍生物及其制备方法和应用,本发明通过将氨基膦酸酯衍生物与康普瑞汀类似物偶联,通过将引入自身具有一定抗癌活性的氨基膦酸酯衍生物引入到CA-4类似物的结构中,对其结构进行优化,可增强其抗肿瘤活性。本发明合成的康普瑞汀衍生物对人多种肿瘤细胞株显示出了较好抗肿瘤活性,其中代表性化合物3e瘤活性优于阳性药物CA-4,且对人正常的肝细胞毒性明显低于CA-4,表明化合物3e潜在靶向性治疗肿瘤性疾病的用途。(The invention discloses a combretastatin derivative and a preparation method and application thereof, the invention couples an aminophosphonate derivative with a combretastatin analogue, and introduces the aminophosphonate derivative with certain anticancer activity into the structure of a CA-4 analogue to optimize the structure, so as to enhance the antitumor activity of the combretastatin derivative. The combretastatin derivative synthesized by the invention shows good anti-tumor activity on various tumor cell strains of human, wherein the tumor activity of a representative compound 3e is superior to that of a positive drug CA-4, and the toxicity on normal liver cells of the human is obviously lower than that of the CA-4, thus indicating the potential targeted application of the compound 3e in treating tumor diseases.)
技术领域
本发明属于药物合成技术领域，涉及一种康普瑞汀衍生物及其制备方法和应用。
背景技术
在医学技术飞速发展的现代，人们已经克服了许多在以往难以治疗的疾病，然而癌症发 病率和致死率还在不断的攀升，已严重威胁人类的生命健康。因此，如何更好的治疗癌症已 经成为现代医药界急需解决的一大难题。目前临床上治疗癌症的手段主要包括化疗、手术治 疗、放疗。由于化疗药物的高效性，因此化疗是大多数癌症患者不可或缺的选择手段之一， 但是其也存在一定缺陷，例如缺乏选择性、严重的毒副作用和耐药。因此，探索具有靶向性 强、疗效高和毒性低的抗癌药物已成为新一代抗癌药物研究中拟解决的关键科学问题。
微管蛋白是由α-微管蛋白和β-微管蛋白的异二聚体形成的蛋白质类多聚物，具有中空管 状结构，在细胞分裂、细胞内物质运输、信号传递、维持细胞形态等过程中起着重要作用。 微管的破坏可诱导细胞周期阻滞在G2/M期，并形成不正常的有丝分裂纺锤体，使其成为抗 肿瘤药物研发的热点靶点之一。康普瑞汀(CA-4)是从天然产物中分离提取得到的，属于微管 蛋白抑制剂类的抗癌药物，目前还处在三期临床研究阶段。研究表明，CA-4对人多种实体瘤 具有优越的抗癌活性，但是其存在毒性较大和水溶性较差等缺点，因此开发具有高效、靶向 和低毒的新型CA-4衍生物具有重要的应用价值和学术意义。
发明内容
针对现有技术的不足，本发明的目的在于提供一种康普瑞汀衍生物，通过将自身具有一 定抗癌活性的氨基膦酸酯衍生物引入到CA-4类似物的结构中，对其结构进行优化，可增强 其抗肿瘤活性。本发明的另一目的在于提供该偶联物的制备方法。
本发明是通过以下技术方案实现的：
一种康普瑞汀衍生物，其结构通式如式I所示：
其中，R1为H、Cl或OCH3，R2为H、F、Cl、Br、CH3或OCH3,n＝1或3。
本发明的进一步改进方案为：
一种康普瑞汀衍生物的制备方法，包括如下步骤：
步骤一，使CA-4与碳酸乙烯酯进行反应得到中间体(1)；
步骤二，使中间体(1)与氨基膦酸脂类衍生物(2)进行偶联反应得到式(Ⅰ)；
反应方程式如下所示：
其中，R1为H、Cl或OCH3，R2为H、F、Cl、Br、CH3或OCH3,n＝1或3。
本发明的更进一步改进方案为：
一种康普瑞汀衍生物的制备方法，包括如下步骤：
步骤一，使CA-4溶于DMF，在无机碱的作用下，在氮气保护下，与碳酸乙烯酯发生酯化反应，得到中间体(1)化合物，其中化合物用量以物质的量计，CA-4：碳酸乙烯酯：无 机碱＝1:(2～4):(2～4)；
步骤二，使氨基膦酸脂类衍生物(2)溶于DCM，在有机碱和催化剂的作用下，与中间体(1)发生偶联反应，得到式(Ⅰ)化合物，其中化合物用量以物质的量计，中间体(1)： 氨基膦酸脂类衍生物(2)：有机碱：催化剂＝1:(1～1.2):(2～2.4):(1.5～1.8)。
本发明的再进一步改进方案为：
步骤一中，所述无机碱为无水碳酸钾，酯化反应的温度为90℃～110℃下回流，反应时 间为10～14h。
步骤二中，所述有机碱为EDCI，所述催化剂为DMAP，偶联反应的温度为-5℃～5℃的冰 浴条件下，反应时间为4～8h。
进一步的，该方法的各个步骤中还包括分离纯化的步骤。
进一步的，上述康普瑞汀衍生物在制备抗肿瘤药物中的应用。
本发明的有益效果为：
本发明利用氨基膦酸酯自身具有抗肿瘤活性的优势，将其引入康普瑞汀类似物结构中可 增强其抗肿瘤活性。
本发明的康普瑞汀衍生物对人多种肿瘤细胞株显示出了较好抗肿瘤活性，其中代表性化 合物3e抗肿瘤活性优于阳性药物CA-4，且对人正常的肝细胞毒性明显低于CA-4，表明化合 物3e具有潜在靶向性治疗肿瘤性疾病的用途。
具体实施方式
本发明提供的康普瑞汀衍生物，其结构通式如式I所示：
合成路线如下：
其中，R1为H、Cl或-OCH3，R2为H、F、Cl、Br、-CH3或-OCH3,n＝1或3。
实施例1～实施例16
实施例1～实施例16中使用的氨基膦酸酯衍生物类化合物2如表1所示，
表1氨基膦酸酯衍生物类化合物2的选择
序号
n
R<sub>1</sub>
R<sub>2</sub>
实施例1(3a)
1
H
H
实施例2(3b)
1
H
F
实施例3(3c)
1
H
Cl
实施例4(3d)
1
H
Br
实施例5(3e)
1
H
OCH<sub>3</sub>
实施例6(3f)
1
H
CH<sub>3</sub>
实施例7(3g)
1
OCH<sub>3</sub>
H
实施例8(3h)
1
Cl
H
实施例9(3i)
3
H
H
实施例10(3j)
3
H
F
实施例11(3k)
3
H
Cl
实施例12(3l)
3
H
Br
实施例13(3m)
3
H
OCH<sub>3</sub>
实施例14(3n)
3
H
CH<sub>3</sub>
实施例15(3o)
3
OCH<sub>3</sub>
H
实施例16(3p)
3
Cl
H
具体制备步骤如下：
(1)中间体1的制备
将CA-4(2.85g,9.0mmol)溶入10mL DMF，加入碳酸乙烯酯(2.38g,27.0mmol)， 最后加入无水碳酸钾(3.72g,27.0mmol)，氮气保护条件下，加热至100℃，回流反应12h. 采用TCL薄层分析法判断反应的终点。反应结束后，向反应物中添加冰水猝灭反应，将反应 物置入125mL分液漏斗中，加入50mL水、50mL DCM和少量NaCl水溶液进行萃取，收 集有机层，重复3次，有机相用无水硫酸钠干燥，旋蒸除去DCM，进行柱层析分离，最终即 得中间体1(1.66g,51.2％)。1H NMR(600MHz,CDCl3)δ6.90(dd,J＝8.3,1.9Hz,1H),6.85(d, J＝2.0Hz,1H),6.77(d,J＝8.3Hz,1H),6.50(s,2H),6.48(d,J＝12.1Hz,1H),6.45(d,J＝12.1 Hz,1H),3.91(t,J＝4.5Hz,2H),3.84-3.83(m,5H),3.82(s,3H),3.70(s,6H).13C NMR(150MHz,CDCl3)δ152.99,148.95,147.47,137.09,132.95,130.08,129.48,129.05,123.00,115.20,111.30, 105.91,71.10,61.11,60.93,55.98,55.86.
(2)中康普瑞汀衍生物的制备
将氨基膦酸酯衍生物类化合物2(150mg,0.321mmol)溶入5mL DCM，加入EDCI(123mg,0.642mmol)和DMAP(59mg,0.482mmol)，最后加入中间体1(100mg,0.289mmol)， 冰浴反应条件下，反应6h。采用TCL薄层分析法判断反应的终点。反应结束后，向反应物 中添加冰水猝灭反应，将反应物置入125mL分液漏斗中，加入50mL水、50mLDCM和少 量NaCl水溶液进行萃取，收集有机层，重复3次，有机相用无水硫酸钠干燥，旋蒸除去DCM， 进行柱层析分离，最终即得康普瑞汀衍生物。
本发明中CA-4根据文献[European Journal ofMedicinal Chemistry,2012,56:166-178]报道 合成。
实施例1～实施例16制得的康普瑞汀衍生物的数据如下：
实施例1：143mg,yield:68.5％.1HNMR(400MHz,CDCl3)δ7.45(d,J＝7.4Hz,2H),7.32 (t,J＝7.4Hz,2H),7.26-7.23(m,1H),6.99(d,J＝8.3Hz,2H),6.91-6.88(m,1H),6.82(d,J＝1.8 Hz,1H),6.76(d,J＝8.3Hz,1H),6.52(d,J＝8.5Hz,2H),6.50(s,2H),6.45(d,J＝2.9Hz,2H), 4.72(d,J＝24.3Hz,1H),4.33-4.31(m,2H),4.13-4.07(m,2H),4.02-4.00(m,2H),3.95-3.84(m, 2H),3.81(s,3H),3.79(s,3H),3.67(s,7H),3.46(s,2H),1.27(t,J＝7.0Hz,3H),1.10(t,J＝7.0 Hz,3H).13C NMR(100MHz,CDCl3)δ171.97,152.99,149.02,147.45,145.44,145.29,137.17, 135.87,132.89,130.06,129.96,129.48,129.02,128.62,127.97,127.94,127.81,127.86,123.49, 122.91,114.94,113.92,111.74,105.97,67.07,63.35,63.28,62.91,60.89,56.86,56.00,55.96, 55.36,40.08,16.47,16.22.HR-MS(m/z)(ESI):calcd for C39H46NO10P[M+H]+:720.2938；found: 720.2946.
实施例2：150mg,yield:70.3％.1HNMR(400MHz,CDCl3)δ7.42-7.41(m,2H),7.06(d,J＝ 19.4Hz,1H),7.00(d,J＝8.5Hz,3H),6.89(d,J＝8.3Hz,1H),6.82(s,1H),6.76(d,J＝8.2Hz, 1H),6.51-6.49(m,3H),6.45(d,J＝1.8Hz,2H),4.70(d,J＝24.2Hz,1H),4.33–4.31(m,2H), 4.13-4.07(m,2H),4.02-3.97(m,2H),3.96-3.86(m,2H),3.81(s,3H),3.80(s,3H),3.67(s,6H), 3.46(s,2H),1.27(t,J＝7.0Hz,3H),1.13(t,J＝7.0Hz,3H).13C NMR(100MHz,CDCl3)δ 171.95,152.99,149.01,147.44,145.23,145.08,137.17,132.88,131.62,130.12,129.96,129.47, 129.40,129.03,123.72,122.92,115.71,115.50,114.92,113.92,111.73,105.97,67.07,63.41, 63.27,62.94,60.88,56.18,56.00,55.95,54.67,40.06,16.46,16.26.HR-MS(m/z)(ESI):calcd for C39H45FNO10P[M+H]+:738.2843；found:738.2872.
实施例3：139mg,yield:63.9％.1HNMR(400MHz,CDCl3)δ7.39(d,J＝8.4Hz,2H),7.29 (d,J＝8.3Hz,2H),7.00(d,J＝8.3Hz,2H),6.91-6.88(m,1H),6.82(d,J＝1.7Hz,1H),6.76(d,J ＝8.3Hz,1H),6.50(s,2H),6.48(d,J＝8.5Hz,2H),6.45(d,J＝2.9Hz,2H),4.69(d,J＝24.4Hz, 1H),4.33-4.31(m,2H),4.15-4.07(m,2H),4.02-3.99(m,2H),3.97-3.86(m,2H),3.81(s,3H),3.80 (s,3H),3.67(s,6H),3.47(s,2H),1.28(t,J＝7.0Hz,3H),1.15(t,J＝7.0Hz,3H).13C NMR(100 MHz,CDCl3)δ171.92,152.99,149.01,147.44,145.15,145.01,137.17,134.58,133.77,132.88, 130.13,129.96,129.47,129.17,129.11,129.03,128.83,123.82,122.93,114.93,113.92,111.74, 105.97,67.08,63.48,63.39,62.94,60.89,56.34,56.00,55.96,54.85,40.06,16.47,16.28.HR-MS (m/z)(ESI):calcdfor C39H45ClNO10P[M+H]+:754.2548；found:754.2587.
实施例4：161mg,yield:69.7％.1HNMR(400MHz,CDCl3)δ7.44(d,J＝6.8Hz,2H),7.32 (d,J＝5.8Hz,2H),6.99(d,J＝7.0Hz,2H),6.89(d,J＝8.0Hz,1H),6.81(d,J＝1.7Hz,1H),6.76 (d,J＝8.1Hz,1H),6.49(s,2H),6.45(d,J＝2.5Hz,2H),6.34-6.26(m,2H),4.66(d,J＝24.4Hz, 1H),4.31-4.25(m,2H),4.14-4.06(m,2H),4.03-3.96(d,J＝3.4Hz,2H),3.94-3.82(m,2H),3.80 (s,3H),3.79(s,3H),3.67(s,6H),3.46(s,2H),1.27(t,J＝7.0Hz,3H),1.12(t,J＝7.0Hz,3H).13C NMR(100MHz,CDCl3)δ171.92,162.63,152.99,149.05,147.44,145.14,144.09,137.17,135.12, 132.89,131.74,130.14,129.97,129.51,129.47,129.03,123.83,122.94,121.91,114.93,113.93, 111.75,105.98,67.08,63.51,63.42,62.94,60.89,56.41,56.01,55.97,54.91,40.06,16.47, 16.29.HR-MS(m/z)(ESI):calcd for C39H45BrNO10P[M+H]+:798.2043；found:798.2067.
实施例5：156mg,yield:72.1％.1HNMR(400MHz,CDCl3)δ7.37-7.34(m,2H),6.99(d,J＝ 8.4Hz,2H),6.90-6.88(m,1H),6.85(d,J＝8.6Hz,2H),6.82(d,J＝1.7Hz,1H),6.76(d,J＝8.3 Hz,1H),6.52(d,J＝8.7Hz,2H),6.49(s,2H),6.45(d,J＝3.0Hz,2H),4.66(d,J＝23.8Hz,1H), 4.33-4.21(m,2H),4.12-4.06(m,2H),4.02-4.00(m,2H),3.94-3.86(m,2H),3.81(s,3H),3.79(s, 3H),3.76(s,3H),3.67(s,6H),3.46(s,2H),1.27(t,J＝7.1Hz,3H),1.12(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ172.03,159.32,152.99,149.01,147.45,145.49,145.34,137.16,132.89, 130.04,129.96,129.48,129.02,128.92,127.62,123.43,122.91,114.93,114.,111.74,105.97, 67.08,63.30,63.23,62.91,60.89,56.15,56.00,55.95,55.23,54.64,40.09,16.48,16.29.HR-MS (m/z)(ESI):calcd forC40H48NO11P[M+H]+:750.3043；found:750.3030.
实施例6：137mg,yield:64.7％.1HNMR(400MHz,CDCl3)δ7.27(d,J＝2.2Hz,2H),7.23 (d,J＝3.7Hz,1H),7.20(d,J＝7.5Hz,1H),7.07(d,J＝6.7Hz,1H),7.01(d,J＝8.2Hz,2H),6.91 (d,J＝8.1Hz,1H),6.84(d,J＝1.2Hz,1H),6.77(d,J＝8.3Hz,1H),6.54(d,J＝8.4Hz,2H),6.51 (s,2H),6.47(d,J＝2.9Hz,2H),4.69(d,J＝24.3Hz,1H),4.35-4.33(m,2H),4.14-4.08(m,2H), 4.04-4.02(m,2H),3.95-3.88(m,2H),3.83(s,3H),3.81(s,3H),3.69(s,6H),3.48(s,2H),2.34(s, 3H),1.29(t,J＝7.0Hz,3H),1.12(t,J＝7.0Hz,3H).13CNMR(100MHz,CDCl3)δ172.00, 152.99,149.01,147.45,145.54,145.40,138.23,137.17,135.75,132.89,130.06,129.96,129.48, 129.02,128.81,128.47,128.41,124.98,123.40,122.91,114.94,113.89,111.74,105.97,67.08, 63.35,63.26,62.92,60.89,56.82,56.00,55.96,55.33,40.09,21.47,16.46,16.20.HR-MS(m/z) (ESI):calcd forC40H48NO10P[M+H]+:734.3094；found:734.3071.
实施例7：155mg,yield:71.3％.1HNMR(400MHz,CDCl3)δ7.25-7.21(m,1H),7.03(d,J＝ 7.6Hz,1H),6.99(d,J＝7.8Hz,2H),6.89(d,J＝8.0Hz,1H),6.81(d,J＝8.9Hz,2H),6.77(d,J＝ 4.3Hz,1H),6.73(d,J＝13.0Hz,1H),6.53(d,J＝8.2Hz,2H),6.49(s,2H),6.45(d,J＝2.5Hz, 2H),4.68(d,J＝24.3Hz,1H),4.33-4.31(m,2H),4.13-4.07(m,2H),4.02-4.00(m,2H),3.95-3.90 (m,2H),3.81(s,3H),3.79(s,3H),3.77(s,3H),3.67(s,6H),3.46(s,2H),1.28(t,J＝7.0Hz,3H), 1.12(t,J＝7.0Hz,3H).13C NMR(100MHz,CDCl3)δ171.98,159.82,152.99,149.02,147.45, 145.49,145.38,137.55,137.16,132.89,130.06,130.06,129.95,129.59,129.48,129.01,123.51, 122.91,120.25,114.94,113.91,113.50,113.42,111.73,105.97,67.08,63.40,63.32,62.92,60.88, 56.90,56.00,55.95,55.40,55.22,40.09,16.47,16.25.HR-MS(m/z)(ESI):calcd for C40H48NO11P [M+H]+:750.3043；found:750.3032.
实施例8：136mg,yield:62.3％.1HNMR(400MHz,CDCl3)δ7.40-7.38(m,2H),7.30(d,J＝ 8.3Hz,2H),7.00(d,J＝8.5Hz,2H),6.91-6.88(m,1H),6.82(d,J＝1.9Hz,1H),6.76(d,J＝8.3 Hz,1H),6.50(s,2H),6.48(s,2H),6.47(d,J＝2.9Hz,2H),6.46(d,J＝2.6Hz,2H),4.69(d,J＝ 24.5Hz,1H),4.34-4.31(m,2H),4.13-4.07(m,2H),4.03-4.01(m,2H),3.99-3.85(m,2H),3.81(s, 3H),3.80(s,3H),3.68(s,6H),3.47(s,2H),1.28(t,J＝7.1Hz,3H),1.15(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ171.92,152.99,149.09,147.63,145.19,145.00,137.17,134.57,133.78, 132.88,130.13,129.96,129.46,129.16,129.11,129.03,128.83,123.82,122.92,114.93,113.92, 111.73,105.97,67.08,63.48,63.39,62.94,60.89,56.34,56.00,55.96,54.84,40.06,16.46,16.28. HR-MS(m/z)(ESI):calcdfor C39H45ClNO10P[M+H]+:754.2548；found:754.2572.
实施例9：159mg,yield:73.5％.1H NMR(400MHz,CDCl3)δ7.45(d,J＝7.4Hz,2H),7.31 (t,J＝7.4Hz,2H),7.26-7.21(m,1H),6.89(s,2H),6.80(d,J＝1.9Hz,1H),6.73(d,J＝8.3Hz, 1H),6.51(s,2H),6.48(d,J＝11.6Hz,2H),6.45(d,J＝3.6Hz,2H),4.72(d,J＝24.2Hz,1H), 4.32-4.29(m,2H),4.15-4.07(m,2H),4.01-3.98(m,2H),3.95-3.89(m,2H),3.81(s,3H),3.76(s, 3H),3.67(s,6H),2.46(t,J＝7.5Hz,2H),2.27(t,J＝7.5Hz,2H),1.86-1.78(m,2H),1.27(t,J＝ 7.1Hz,3H),1.10(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ173.44,152.98,148.97, 147.43,144.55,144.40,137.16,136.03,132.89,131.24,129.90,129.49,129.19,128.99,128.59, 127.89,127.83,122.86,114.76,113.95,111.63,105.97,67.07,63.30,63.23,62.49,60.88,57.02, 55.96,55.91,55.52,34.14,33.44,26.66,16.46,16.22.HR-MS(m/z)(ESI):calcd for C41H50NO10P [M+H]+:748.3251；found:748.3243.
实施例10：155mg,yield:70.1％.1H NMR(400MHz,CDCl3)δ7.44-7.41(m,2H),7.01(t,J ＝8.5Hz,2H),6.89(d,J＝7.9Hz,3H),6.82(s,1H),6.74(d,J＝8.3Hz,1H),6.50(s,2H),6.48(d, J＝11.6Hz,2H),6.45(d,J＝2.9Hz,2H),4.70(d,J＝24.1Hz,1H),4.32-4.30(m,2H),4.13-4.07 (m,2H),4.01-3.99(m,2H),3.97-3.86(m,2H),3.81(s,3H),3.77(s,3H),3.68(s,6H),2.47(t,J＝ 7.4Hz,2H),2.28(t,J＝7.5Hz,2H),1.86-1.79(m,2H),1.27(t,J＝7.0Hz,3H),1.14(t,J＝7.0Hz, 3H).13C NMR(100MHz,CDCl3)δ173.44,152.99,147.54,144.33,144.18,137.17,132.89, 131.79,131.47,129.91,129.50,129.44,129.36,129.24,129.00,122.87,115.68,115.46,114.76, 113.95,111.62,105.97,67.08,63.40,63.28,62.50,60.88,56.35,55.96,55.91,54.84,34.13,33.42, 26.63,16.46,16.31.HR-MS(m/z)(ESI):calcd for C41H59FNO10P[M+H]+:766.3156；found: 766.3166.
实施例11：142mg,yield:62.7％.1H NMR(400MHz,CDCl3)δ7.40-7.38(m,2H),7.29(d,J ＝8.3Hz,2H),6.90-6.87(m,3H),6.82(d,J＝1.8Hz,1H),6.74(d,J＝8.3Hz,1H),6.50(s,2H), 6.48(d,J＝2.4Hz,2H),6.45(d,J＝3.5Hz,2H),4.69(d,J＝24.3Hz,1H),4.32-4.30(m,2H), 4.14-4.07(m,2H),4.01-3.94(m,2H),4.01-3.99(m,2H),3.81(s,3H),3.77(s,3H),3.68(s,6H), 2.47(t,J＝7.5Hz,2H),2.28(t,J＝7.5Hz,2H),1.87-1.79(m,2H),1.27(t,J＝7.1Hz,3H),1.15(t, J＝7.0Hz,3H).13C NMR(100MHz,CDCl3)δ173.43,152.98,148.96,147.39,144.32,144.09, 137.16,134.73,133.70,132.89,131.56,129.90,129.48,129.25,129.19,129.00,128.79,122.87, 114.75,113.95,111.62,105.97,67.07,63.49,63.34,62.50,60.88,56.49,55.96,55.91,55.00,34.13, 33.42,26.63,16.46,16.27.HR-MS(m/z)(ESI):calcd for C41H59ClNO10P[M+H]+:782.2861；found: 782.2865.
实施例12：166mg,yield:69.4％.1H NMR(400MHz,CDCl3)δ7.45(d,J＝8.3Hz,2H),7.35 (d,J＝2.2Hz,1H),7.33(d,J＝2.2Hz,1H),6.91-6.88(m,3H),6.83(d,J＝1.8Hz,1H),6.75(d,J ＝8.3Hz,1H),6.50(s,2H),6.48(d,J＝4.4Hz,2H),6.45(d,J＝2.9Hz,2H),4.67(d,J＝24.4Hz, 1H),4.33-4.30(m,2H),4.16-4.07(m,2H),4.02-4.00(m,2H),3.97-3.86(m,2H),3.82(s,3H),3.78 (s,3H),3.68(s,6H),2.47(t,J＝7.5Hz,2H),2.29(t,J＝7.5Hz,2H),1.87-1.80(m,2H),1.28(t,J ＝7.1Hz,3H),1.16(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ173.43,152.99,148.96, 147.43,144.23,144.08,137.17,135.27,132.89,131.74,131.58,129.91,129.53,129.48,129.26, 129.00,122.88,121.84,114.76,114.00,111.62,105.97,67.08,63.49,63.45,62.51,60.89,56.58, 55.97,55.92,55.08,34.13,33.43,26.64,16.47,16.29.HR-MS(m/z)(ESI):calcd for C41H59BrNO10P[M+H]+:826.2356；found:826.2393.
实施例13：165mg,yield:73.3％.1H NMR(400MHz,CDCl3)δ7.37(d,J＝2.1Hz,1H),7.35 (d,J＝2.2Hz,1H),6.88(d,J＝8.4Hz,3H),6.85(d,J＝8.6Hz,2H),6.82(d,J＝1.7Hz,1H),6.74 (d,J＝8.3Hz,1H),6.51(d,J＝2.0Hz,1H),6.50(s,2H),6.48(d,J＝8.0Hz,1H),6.45(d,J＝3.6 Hz,2H),4.67(d,J＝23.8Hz,1H),4.32-4.30(m,2H),4.12-4.07(m,2H),4.01-3.99(m,2H), 3.97-3.86(m,2H),3.81(s,3H),3.76(s,3H),3.76(s,3H),3.68(s,6H),2.46(t,J＝7.5Hz,2H), 2.28(t,J＝7.5Hz,2H),1.86-1.79(m,2H),1.27(t,J＝7.1Hz,3H),1.13(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ173.45,159.27,152.98,148.97,147.43,144.59,144.44,137.16,132.89, 131.19,129.90,129.48,129.17,128.99,128.93,127.80,122.86,114.78,114.05,113.98,111.62, 105.97,67.08,63.24,63.17,62.49,60.88,56.32,55.95,55.91,55.21,54.81,34.15,33.45,26.67, 16.48,16.29.HR-MS(m/z)(ESI):calcd for C42H52NO11P[M+H]+:778.3356；found:778.3396.
实施例14：138mg,yield:62.7％.1H NMR(400MHz,CDCl3)δ7.34(d,J＝1.9Hz,1H),7.32 (d,J＝2.0Hz,1H),7.12(d,J＝7.8Hz,2H),6.89-6.84(d,J＝8.2Hz,3H),6.82(d,J＝3.6Hz,1H), 6.73(d,J＝8.3Hz,1H),6.51(d,J＝1.4Hz,2H),6.50(s,2H),6.45(d,J＝3.6Hz,2H),4.68(d,J＝ 24.0Hz,1H),4.32-4.30(m,2H),4.13-4.07(m,2H),4.01-3.99(m,2H),3.96-3.86(m,2H),3.81(s, 3H),3.76(s,3H),3.67(s,6H),2.46(t,J＝7.5Hz,2H),2.31(s,3H),2.27(t,J＝7.5Hz,2H), 1.86-1.78(m,2H),1.27(t,J＝7.1Hz,3H),1.12(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ 173.53,153.13,148.97,147.51,144.62,144.67,137.57,137.16,132.90,131.17,129.90,129.49, 129.32,129.17,128.99,127.76,127.64,122.93,114.83,113.96,111.58,105.97,67.08,63.26, 63.18,62.49,60.89,56.71,55.96,55.91,55.21,34.15,33.46,26.67,21.14,16.47,16.26.HR-MS (m/z)(ESI):calcd for C42H52NO10P[M+H]+:762.3407；found:762.3437.
实施例15：155mg,yield:68.9％.1H NMR(400MHz,CDCl3)δ7.22(t,J＝7.9Hz,1H),7.05-7.01(m,2H),6.88(d,J＝8.1Hz,3H),6.82(s,1H),6.78(d,J＝8.3Hz,1H),6.73(d,J＝8.3 Hz,1H),6.51(d,J＝8.2Hz,2H),6.49(s,2H),6.45(d,J＝3.7Hz,2H),4.69(d,J＝24.2Hz,1H), 4.32-4.31(m,2H),4.13-4.07(m,2H),4.01-3.99(m,2H),3.96-3.85(m,2H),3.81(s,3H),3.76(s, 3H),3.73(s,3H),3.67(s,6H),2.46(t,J＝7.4Hz,2H),2.28(t,J＝7.4Hz,2H),1.86-1.79(m,2H), 1.27(t,J＝7.0Hz,3H),1.12(t,J＝7.0Hz,3H).13C NMR(100MHz,CDCl3)δ173.45,159.79, 152.98,148.97,147.43,144.60,144.45,137.72,137.16,132.93,131.27,129.89,129.56,129.49, 129.19,128.99,122.86,120.29,114.77,113.94,113.46,113.37,111.62,105.97,67.08,63.33, 63.26,62.49,60.88,57.07,55.96,55.90,55.58,55.22,34.15,33.46,26.68,16.47,16.25.HR-MS (m/z)(ESI):calcd for C42H52NO11P[M+H]+:778.3356；found:778.3320.
实施例16：167mg,yield:73.7％.1H NMR(400MHz,CDCl3)δ7.43(d,J＝2.3Hz,1H),7.35 (d,J＝6.9Hz,1H),7.28-7.22(m,2H),6.91(d,J＝8.2Hz,2H),6.88(d,J＝1.6Hz,1H),6.83(d,J ＝1.5Hz,1H),6.74(d,J＝8.3Hz,1H),6.50(s,2H),6.49(d,J＝8.7Hz,2H),6.45(d,J＝3.6Hz, 2H),4.69(d,J＝24.5Hz,1H),4.33-4.21(m,2H),4.14-4.09(m,2H),4.02-3.97(m,2H),3.82(s, 3H),3.77(s,3H),3.68(s,6H),2.48(t,J＝7.5Hz,2H),2.29(t,J＝7.4Hz,2H),1.88-1.80(m,2H), 1.28(t,J＝7.1Hz,3H),1.15(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ173.43,152.98, 148.96,147.37,144.31,144.09,138.50,137.16,134.53,132.89,131.59,129.90,129.85,129.49, 129.28,128.99,128.13,127.96,126.01,122.87,114.75,113.90,111.62,105.97,67.07,63.50, 63.41,62.50,60.88,56.70,55.96,55.90,55.21,34.13,33.44,26.65,16.45,16.22.HR-MS(m/z) (ESI):calcdfor C41H59ClNO10P[M+H]+:782.2861；found:782.2856.
测试例1
实施例1～实施例16制得的化合物3a-3p体外抗肿瘤活性研究。
为了探究本发明所制备的化合物3a-3p对癌细胞是否具有较好的杀伤作用，用MTT法实 验对其进行了研究，其结果见表2。从表2中可以看出化合物3a-3p对HepG-2(肝癌)、HT29 (肠癌)、A549(肺癌)和MGC-803(胃癌)均有较好的毒活性，一些化合物如3e、3g和 3l表现出良好的抗癌活性，其IC50值均小于1μM，其中化合物3e的抗癌活性明显优于其他 化合物和阳性药物CA-4，对癌细胞HepG-2的IC50值为0.36μM；对癌细胞HT29的IC50值 为0.31μM；对癌细胞A549的IC50值为0.19μM；对癌细胞MGC-803的IC50值为0.42μM； 对目标化合物结构进行分析可以发现，在化合物3i-3p中，相比苯环C-4位被卤素取代的化 合物，被-OCH3和-CH3取代化合物3e和3f具有更好的体外抗肿瘤活性，在化合物3i-3p中， 也存在这样的现象，氨基膦酸脂的苯环C-4位被-OCH3和-CH3取代的3l和3m也比被卤素取 代的3j、3h和3k的活性更好，表明在苯环的对位上引入-OCH3和-CH3是一个正确的选择。 综合比较，化合物3e的抗癌活性明显优于其他化合物，与阳性对照药物康普瑞汀相比活性更 好。
表2.化合物3a-3p对所测试的人癌细胞株的IC50值。
测试例2
实施例1～实施例16制得的化合物3a-3p对人正常肝细胞的毒性研究。
为了探究本发明所制备的化合物3a-3p对人正常细胞的毒性，用MTT法实验对其进行了 测试，其结果见表3。根据表3可以发现目标化合物3a-3p对人正常肝细胞的毒性低于阳性药 物CA-4(IC50＝3.27±0.82μM)低，其IC50值的范围在9.33～20.14μM。另外，其抗癌活性 最优的化合物3e(IC50＝10.45±0.98μM)对人正常肝细胞的毒性明显低于阳性药物，该实验 结果也进一步表明，将氨基膦酸酯衍生物引入到CA-4的骨架中，不仅可以提高抗癌活性， 同时也能降低化合物对正常细胞的毒性，表明化合物对癌细胞具有较好的选择性。
表3.化合物3a-3p对人正常肝细胞LO2的IC50值。